Method for constructing a chimeric dna library using a single strand speific dnase
a dna library and single-strand technology, applied in the field of constructing a chimeric dna library, can solve the problems of high labor intensity, high cost, and ineffective above methods in the reassembly of heterologous dna fragments with low sequence similarity, and achieve the effect of enhancing the production of industrially useful proteins
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example 1
Preparation of Template DNA
[0060] To test the inventive method, the construction of a chimeric DNA library was carried out through reassembly of heterologous DNA of chitinase genes.
[0061] The total nucleic acid of each of Aeromonas hydrophila (KCTC 2358), Pantoea agglomerans (KCTC 2578) and Aeromonas punctata (KCTC 2944) was purified using a genome DNA prep kit (Promega). Each chitinase gene was amplified using the purified nucleic acid as a template and Chi600f of SEQ ID NO. 1 and Chi1200r of SEQ ID NO. 2 as a primer pair. PCR was performed by using Vent polymerase (NEB) and Taq polymerase (Bioneer) together. The reaction procedure consisted of a primary denaturation step at 94° C. for 3 min, 30 cycles of a denaturation step at 94° C. for 30 sec, an annealing step at 50° C. for 30 sec, an extension step at 72° C. for 30 sec, and an further extension step at 72° C. for 30 min. Amplified PCR product was cloned into T-vector (Promega) and subjected to sequence analysis.
[0062] The r...
example 2
Reassembly Experiment 1
(2-1) Hybridization of Heterologous DNA Sequences
[0064] Heterologous DNA sequences used for a reassembly experiment were prepared by digesting a plasmid containing Pantoea agglomerans (KCTC 2578) or Aeromonas hydrophila (KCTC 2358) chitinase gene with restriction enzymes SacII and SpeI and extracting a chitinase gene region from agarose gel. 0.2 μg each of the DNAs were mixed with S1 nuclease buffer (30 mM sodium acetate, 1 mM zinc acetate, 5% (v / v) glycerol) and 300 mM NaCl to a final volume of 30 μl, incubated at 95° C. for 10 min, and then, gradually cooled to induce DNA hybridization.
(2-2) Preparation of Internal Primers
[0065] S1 nuclease was added to the reaction mixture of Example (2-1) to a final concentration of 1,000 units / μl, and incubated at 45° C. for 1 hour, to cleave single strand DNA regions which did not form complementary bonds. Double strand DNA fragments formed between matching sequence regions of the two heterologous chitinase genes w...
example 3
Use of Restriction Enzyme-Digested DNA for Hybridization Reaction (Reassembly Experiment 2)
(3-1) Hybridization of Heterologous DNA Sequences and Preparation of Internal Primers
[0070] Chitinase genes of Pantoea agglomerans (KCTC 2578) and Aeromonas hydrophila (KCTC 2358) prepared in Example 1 were subjected to amplify using Chi600f primer of SEQ ID NO. 1 and Chi1200r primer of SEQ ID NO. 2. 0.5 μg each of the chitinase genes were mixed with S1 nuclease buffer (30 mM sodium acetate, 1 mM zinc acetate, 5% (v / v) glycerol) and 300 mM NaCl to a final volume of 20 μl, incubated at 95° C. for 10 min, and gradually cooled to induce DNA hybridization.
[0071] Employed in the above procedure were full-length Aeromonas hydrophila DNA amplified by using Chi600f and Chi1200r primer pair, and HpaII-digested DNA fragment of Pantoea agglomerans gene.
[0072] The preparation of complementary internal primers by treating with S1 nuclease was performed as in Example (2-2).
(3-2) DNA Reassembly Using ...
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